Summary A central problem in biology is to understand how cells optimize biological processes in response to changing environmental conditions. mRNA decay mechanisms contribute to coordination of activities by limiting the number of times each mRNA can be translated into protein molecules. Recent studies have identified a novel 5'-end-dependent mRNA decay pathway in bacteria triggered by the initial conversion of the triphosphorylated 5' end of primary transcripts to a monophosphate by the Nudix hydrolase RppH. A triphosphate on the 5'-end of bacterial mRNA has a protective effect analogous to that of the cap structure of eukaryotic mRNAs; therefore, pyrophosphate removal by RppH parallels the action of eukaryotic decapping enzymes. Despite its vital importance, 5'-end-dependent mRNA decay is among the least understood mechanisms of gene regulation. This proposal details a set of specific aims that address specificity, catalytic mechanism, and the role of RppH and RppH-associated factors in regulating gene expression in bacteria. The proposal integrates X-ray crystallography, genetics, biochemical methods, focused genome-wide RNA sequencing and bioinformatics. Specific Aim 1 is devoted to the determination of the catalytic mechanism of RppH and understanding the substrate specificity of RppH. This Aim will address molecular principles of RNA `decapping' based on the crystal structures of Escherichia coli RppH bound to model RNAs and structure-guided mutagenesis. Specific Aim 2 will validate a new model for the 5'-end-dependent mRNA decay. This Aim will identify and characterize new factors involved in the pathway and their impact on gene expression landscape. Specific Aim 3 will identify cellular mRNA targets of RppH on a genome-wide scale under various environmental conditions. This Aim will reveal the molecular features of RNAs that are important for RppH recognition and uncover the contribution of RppH to the control of gene expression in bacteria. The results of these studies will provide a comprehensive picture of the critical steps in the 5'-end-dependent pathway for bacterial mRNA decay and thereby address a fundamental gap in our understanding of the regulatory networks controlled by RNA degradation.